A nanocompartment system contributes to defense against oxidative stress in Mycobacterium tuberculosis

  1. Katie A Lien
  2. Kayla Dinshaw
  3. Robert J Nichols
  4. Caleb Cassidy-Amstutz
  5. Matthew Knight
  6. Rahul Singh
  7. Lindsay D Eltis
  8. David F Savage
  9. Sarah A Stanley  Is a corresponding author
  1. Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, United States
  2. Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, United States
  3. Department of Plant and Microbial Biology, University of California, Berkeley, United States
  4. Department of Microbiology and Immunology, The University of British Columbia, Canada
  5. School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, United States
4 figures, 1 table and 20 additional files

Figures

Figure 1 with 1 supplement
Mtb produces endogenous nanocompartments that package a peroxidase.

(A) Schematic of the nanocompartment operon in Mtb that encodes the encapsulin shell protein (Cfp29) and the dye-decoloring peroxidase cargo protein (DyP). (B) Transmission electron microscopy (TEM) …

Figure 1—figure supplement 1
Nanocompartment purification and complementation strategies.

(A) Coomassie-stained SDS-PAGE of fractions collected during purification of nanocompartments heterologously expressed in E. coli: (1) ultracentrifugation pellet post-CellLytic B solubilization, (2) …

Nanocompartments protect Mtb from oxidative stress in acidic environments.

(A) OD600 measurements of wild-type and DyP::Tn Mtb grown in 7H9 medium following exposure to H2O2 for 96 hr. Values reported are normalized to the untreated controls. CFU enumeration of wild-type …

Susceptibility of Mtb nanocompartment mutants to oxidative and acid stress is mediated by free fatty acids.

(A) Transposon sequencing (Tn-seq) data showing normalized sequence reads per gene for all putative Mtb peroxidases, catalases, and superoxide dismutases and (B) lipid and cholesterol metabolism Mtb …

Nanocompartment mutants are attenuated for survival in macrophages and are more susceptible to pyrazinamide (PZA) treatment.

CFU enumeration of wild-type Mtb and (A) Δoperon or (B) DyP::Tn mutants during infection of murine bone marrow-derived macrophages. Macrophages were infected with a bacterial MOI of 1, and CFUs were …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mycobacterium tuberculosis)DypGenBankGene ID: 885388, Rv0799c
Gene (My. tuberculosis)Cfp29GenBankGene ID: 885460, Rv0798c
Strain, strain background (Mus musculus)BALB/CThe Jackson LaboratoryStock no: 000651
Strain, strain background (M. tuberculosis)H37RvEric Rubin Lab, Harvard School of Public Health
Strain, strain background (Escherichia coli)BL21 (DE3) LOBSTRkerafastCat# EC1002
Genetic reagent (M. tuberculosis)DyP::TnBroad Institute, Hung LabRv0799c::Tn
Genetic reagent (M. tuberculosis)ΔoperonThis studyΔRv0799c-Rv0798cSee Materials and methods
Genetic reagent (M. tuberculosis)ΔCfp29This studyΔRv0798cSee Materials and methods
Genetic reagent (M. tuberculosis)ΔDyPThis studyΔRv0799cSee Materials and methods
Recombinant DNA reagentPet14bNovagenCat# 69660-3
Recombinant DNA reagentpUV15tetORmAddgeneCat# 17975AHT-inducible
construct for all
complementation
Recombinant DNA reagentpKL4This studyRv0798c
cloned into
pUV15tetORm
(with KanR)
Recombinant DNA reagentpKL5This studyRv0799c
cloned into
pUV15tetORm
(with KanR)
Recombinant DNA reagentpKL6This studyOperon
cloned into
pUV15tetORm
(with KanR)
Recombinant DNA reagentpKL14This studyOperon
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpKL15This studyRv0798c
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpKL16This studyRv0799c
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpUV15 pHGFP HygR:AddgeneCat# 70045Rv0799c
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpMV762-mrx1-roGFP2Amit Singh, ICGEB, IndiaPMC3907381
AntibodyAnti-Mtb Cfp29Rabbit polyclonalProduced by
GenScript USA,
see Materials
and methods
1:10,000
AntibodyHRPGoat anti-rabbit polyclonalSanta Cruz
Biotechnology
sc-2030
1:5000
Chemical compound, drug3-Ethylbenzothia
zoline-6-sulfonic acid
Millipore SigmaCat# 10102946001

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/74358/elife-74358-transrepform1-v2.docx
Supplementary file 1

Mtb transposon sequencing screen in acidified broth with perxoide stress.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp1-v2.xlsx
Source data 1

Original scanned image of the gel depicting Figure 1—figure supplement 1A.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp2-v2.zip
Source data 2

Scanned image of the gel depicting Figure 1—figure supplement 1A.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp3-v2.zip
Source data 3

Original scanned image of the gel depicting Figure 1—figure supplement 1B.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp4-v2.zip
Source data 4

Original scanned image of the gel depicting Figure 1—figure supplement 1B.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp5-v2.zip
Source data 5

Original scanned image of the gel depicting Figure 1—figure supplement 1C.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp6-v2.zip
Source data 6

Original image capture of PVDF membrane to show the molecular weight ladder (Figure 1—figure supplement 1D).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp7-v2.zip
Source data 7

Original fluorescence image of western blot with Ag85B antibody (Figure 1—figure supplement 1D).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp8-v2.zip
Source data 8

Original fluorescence image of western blot with Cfp29 antibody (Figure 1—figure supplement 1D).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp9-v2.zip
Source data 9

Western images from Figure 1—figure supplement 1D.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp10-v2.zip
Source data 10

Original scanned image of the gel depicting both Tn::DypB+DypB and Tn::DypB+Cfp29 (Figure 1—figure supplement 1E).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp11-v2.zip
Source data 11

Original scanned image of the gel depicting Tn::DypB+Operon (Figure 1—figure supplement 1E).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp12-v2.zip
Source data 12

This image depicts Tn::DypB+DypB, Tn::DypB+Cfp29, and Tn::DypB+Operon with molecular weight markers, lane labels, and the section of the image that is depicted in the main figure outlined in blue (Figure 1—figure supplement 1E).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp13-v2.zip
Source data 13

Wig file for control (library only) replicate 1 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp14-v2.zip
Source data 14

Wig file for control (library only) replicate 2 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp15-v2.zip
Source data 15

Wig file for control (library only) replicate 3 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp16-v2.zip
Source data 16

Wig file for experimental (H2O2_low pH) replicate 1 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp17-v2.zip
Source data 17

Wig file for experimental (H2O2_low pH) replicate 2 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp18-v2.zip
Source data 18

Wig file for experimental (H2O2_low pH) replicate 3 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp19-v2.zip

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